Transport of folate compounds by leukemic cells. Evidence for a single influx carrier for methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblasts

Influx kinetics and inhibitor specificity have been compared for the transport of methotrexate, 5-methyltetrahydrofolate, and folate in CCRF-CEM human lymphoblastoid cells. Influx of each folate compound proceeded with approximately the same Vmax, fluctuated in the same fashion with the ionic composition of the medium, and was blocked by low concentrations of an N-hydroxysuccinimide ester of methotrexate in both an anion-deficient buffer and in a buffered saline medium containing physiological concentrations of glucose and bicarbonate. Moreover, methotrexate influx was inhibited by 5-methyltetrahydrofolate and folate, and the inhibition constants (Ki) of the latter compounds were equivalent to their Kt values for half-maximal influx. Folate influx was likewise inhibited by methotrexate. The Ki for methotrexate was equivalent to its Kt for influx, and o-phthalate and phosphate each inhibited folate and methotrexate with the same degree of effectiveness. Various reversible and irreversible inhibitors reduced the influx of each folate substrate by greater than 90%, and the progression of inhibition in each case was indicative of a single uptake component. Folate influx exhibited the same high sensitivity to inhibitors of methotrexate influx when measurements were performed at folate concentrations near the Kt for influx (10-50 microM) or at concentrations approximating physiological conditions (5-20 nM). These results indicate that CCRF-CEM cells possess a single shared transport system for the uptake of methotrexate, 5-methyltetrahydrofolate, and folate and that other high- or low-affinity uptake processes are not present in these cells.     

Characterization of folate transport mediated by a low pH route in mouse L1210 leukemia cells with defective reduced folate carrier function

Folate influx at low pH was characterized in MTXrA cells, an L1210 mouse leukemia cell line with a functional defect in the reduced folate carrier. Folic acid influx in MTXrA cells was negligible at pH 7.5, increased 13-fold as the pH was decreased to 6.0, and was indistinguishable from that in L1210 cells. In contrast, while methotrexate (MTX) influx in MTXrA cells at pH 6.0 was 15-fold higher than at pH 7.5, in L1210 cells it was decreased by half. Influx of MTX, folic acid, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate in MTXrA cells was increased at pH < 7.0, but their pH optima and profile differed substantially. Influx of MTX and 5-methyltetrahydrofolate at pH 6.0 showed saturability, with a Kt of 2.65 and 0.56 microM, and a Vmax of 0.45 and 0.083 nmol/g dry wt/min, respectively. MTX influx mediated by the low pH transporter was insensitive to the anionic composition of the transport buffer and affected minimally (approximately 20%) by Na+ substitution. The anion transport inhibitors sulfobromophthalein, diisothiocyanatostilbene disulfonic acid, and acetamidoisothiocyanatostilbene disulfonic acid were not effective inhibitors of the low pH route. MTX transport at low pH did not increase in MTXrA-R16 cells, an MTXrA derivative with 10-fold overexpression of the reduced folate carrier (RFC) due to transfection with RFC1 cDNA. Inhibition of reduced folate carrier activity with acetamidoisothiocyanatostilbene disulfonic acid resulted in identical MTX influx in L1210, MTXrA, and MTXrA-R16 cells at pH 5.5. Finally, low pH-mediated MTX influx was reduced by energy inhibitors and partially inhibited by ionophores (nigericin > monensin > valinomycin). The data indicate that L1210 and MTXrA cells express similar activities of a low pH folate transporter that has properties distinct from, and independent of, the reduced folate carrier.     

Inhibition of nucleoside and nucleobase transport and nitrobenzylthioinosine binding by dilazep and hexobendine

The transport of 500 microM uridine by human erythrocytes and S49, P388 and L1210 mouse leukemia cells, Chinese hamster ovary (CHO) cells and Novikoff rat hepatoma cells was inhibited strongly by dilazep and hexobendine with similar concentration dependence, but the sensitivity of transport in the various cell types varied greatly; IC50 values ranged from 5-30 nM for human erythrocytes and S49 and P388 cells to greater than 1 microM for CHO and Novikoff cells. The binding of nitrobenzylthioinosine (NBTI) to high-affinity sites on these cells (Kd approximately equal to 0.5 nM) was inhibited by hexobendine and dilazep in a similar pattern. Furthermore, these drugs, just as dipyridamole and papaverine, inhibited the dissociation of NBTI from high-affinity binding sites but only at concentrations 10-100 times higher than those inhibiting uridine transport. In contrast, high uridine concentrations (greater than 2 mM) accelerated the dissociation of NBTI. Dilazep also inhibited the transport of hypoxanthine, but only in those cell lines whose transporter is sensitive to inhibition by uridine and dipyridamole. Adenine transport was not inhibited significantly by dilazep in any of the cell lines tested, except for a slight inhibition in Novikoff cells. [14C]Hexobendine equilibrated across the plasma membrane in human erythrocytes within 2 sec of incubation at 25 degrees, but accumulated to 6-10 times the extracellular concentration in cells of the various cultured lines. Uptake was not affected by high concentrations of uridine, NBTI or dipyridamole. Hexobendine inhibited the growth of various cell lines to a lesser extent (IC50 = greater than or equal to 100 microM) than dipyridamole (IC50 = 15-40 microM). At 40 microM, however, it completely inhibited the growth of S49 cells that had been made nucleoside dependent by treatment with methotrexate or pyrazofurin.     

Kinetics of the interaction of sertraline with the human platelet plasma membrane 5-hydroxytryptamine carrier

Sertraline, a new selective 5-HT uptake inhibitor showed a mixed pattern of inhibition of human platelet 5-HT uptake with a Ki value of 2.5 nM and K'i value of 25 nM. Imipramine and alaproclate were found to be fully competitive inhibitors of 5-HT uptake with Ki values of 8 and 130 nM respectively. Sertraline was a fully competitive inhibitor of high-affinity [3H]imipramine binding to platelet membranes with a Ki value of 1.3 nM, as was alaproclate and 5-HT with Ki values of 170 and 800 nM respectively. Both sertraline and imipramine, at a concentration of 10 microM caused a fast monophasic dissociation of [3H]imipramine from platelet membranes in contrast to serotonin which caused a slow monophasic dissociation.     

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.     

Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia. Interaction with tubulin and effects of cellular microtubules.

Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. We have compared dolastatin 15 with dolastatin 10 for its effects on cells grown in culture and on biochemical properties of tubulin. The IC50 values for cell growth were obtained for dolastatin 15 with L1210 murine leukemia cells, human Burkitt lymphoma cells, and Chinese hamster ovary (CHO) cells (3, 3, and 5 nM with the three cell lines, respectively). For dolastatin 10, IC50 values of 0.4 and 0.5 nM were obtained with the L1210 and CHO cells, respectively. At toxic concentrations dolastatin 15 caused the leukemia and lymphoma cells to arrest in mitosis. In the CHO cells both dolastatin 15 and dolastatin 10 caused moderate loss of microtubules at the IC50 values and complete disappearance of microtubules at concentrations 10-fold higher. Despite its potency and the loss of microtubules in treated cells, the interaction of dolastatin 15 with tubulin in vitro was weak. Its IC50 value for inhibition of glutamate-induced polymerization of tubulin was 23 microM, as compared to values of 1.2 microM for dolastatin 10 and 1.5 microM for vinblastine. Dolastatin 10 noncompetitively inhibits the binding of vincristine to tubulin, inhibits nucleotide exchange, stabilizes the colchicine binding activity of tubulin, and inhibits tubulin-dependent GTP hydrolysis (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990; Bai et al. J Biol Chem 265: 17141-17149, 1990). Only the latter reaction was inhibited by dolastatin 15. Nevertheless, its structural similarity to dolastatin 10 indicates that dolastatin 15 may bind weakly in the "vinca domain" of tubulin (a region of the protein we postulate to be physically close to but not identical with the specific binding site of vinca alkaloids and maytansinoids), presumably in the same site as dolastatin 10 (the "peptide site").     

Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates.

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.     

Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia (Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, Schmidt JM, Baczynskyj L, Tomer KB and Bontems RJ, J Am Chem Soc 109: 6883-6885, 1987). Since our preliminary studies demonstrated that dolastatin 10 inhibited tubulin polymerization and the binding of radiolabeled vinblastine to tubulin, an initial characterization of the properties of dolastatin 10 included a comparison to other antimitotic drugs interfering with vinca alkaloid binding to tubulin (vinblastine, maytansine, rhizoxin, and phomopsin A). Dolastatin 10 inhibited the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 microM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 microM for dolastatin 10, 1.4 microM for phomopsin A, 1.5 microM for vinblastine, 3.5 microM for maytansine, and 6.8 microM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule-associated proteins. Preliminary studies indicated that dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. We confirmed that rhizoxin, phomopsin A, and maytansine also inhibit the binding of radiolabeled vinblastine and vincristine to tubulin. Dolastatin 10 and phomopsin A were the strongest inhibitors of these reactions, and rhizoxin the weakest. Dolastatin 10, phomopsin A, maytansine, vinblastine, and rhizoxin all inhibited tubulin-dependent GTP hydrolysis. The greatest inhibition of hydrolysis was observed with dolastatin 10 and phomopsin A, and the least inhibition with rhizoxin.     

Tumor cell adherence to cultured capillary endothelial cells is promoted by activators of protein kinase C

Stimulation of cells with protein kinase C (PKC)-specific activators such as phorbol esters increased in a reversible manner the rate of adherence of [3H]leucine-labelled L1210 cells to cultured bovine cerebral cortex capillary endothelial cells (CEC). This effect was not specific for L1210 cells since 12-O-tetradecanoyl phorbol 13-acetate (TPA) strongly increased the binding of various other tumor cell lines. Phorbol esters increased the rate of L1210 cell adhesion to CEC by enhancing their binding capacity without affecting the apparent affinity of L1210 cells for CEC. This stimulation was specific to the phorbol analogs which activate PKC since it was not effected by 4 alpha-phorbol didecanoate, known to be inactive for PKC. Down-regulation experiments showed that adhesion enhancement was entirely attributable to an effect on tumor cells without contribution of CEC intracellular PKC. PKC inhibitors like staurosporine, sphingosine and H-7 showed strong antagonistic activity towards TPA-induced L1210 cell adherence to CEC (IC50 = 0.5 nM, 160 nM and 10 microM, respectively). Adhesive proteins such as vitronectin, fibrinogen, fibronectin and the tetrapeptide RGDS, an active sequence from their cell-binding domains, exhibited potent, dose-dependent inhibition of PKC-induced tumor cell adhesion.     

Compartmentation of intracellular folates. Failure to interconvert tetrahydrofolate cofactors to dihydrofolate in mitochondria of L1210 leukemia cells treated with trimetrexate

Following exposure of L1210 leukemia cells to antifolates, tetrahydrofolate-dependent purine and pyrimidine biosyntheses are blocked despite the presence of the major portion of tetrahydrofolate cofactors. Previous studies from this laboratory demonstrated that this cannot be due to direct inhibition of thymidylate synthase by dihydrofolate polyglutamates or other endogenous folates and suggested that this phenomenon is due to compartmentation of tetrahydrofolate cofactors unavailable for interconversion and/or oxidation when dihydrofolate reductase activity is abolished by antifolates. The present paper evaluates the possibility that tetrahydrofolate cofactors in subcellular organelles, in particular, mitochondria, are unavailable for oxidation by thymidylate synthase. Particulate and cytosolic fractions were obtained from L1210 cells following homogenization and differential centrifugation. The crude mitochondrial fraction contained 20.1% of the total folate pool and included 5-formyltetrahydrofolate, 10-formyltetrahydrofolate and tetrahydrofolate in proportions similar to intact cells. The cytosolic fraction had an increased proportion of tetrahydrofolate and decreased proportions of 5-formyl- and 10-formyltetrahydrofolate relative to intact cells or the particulate fraction. Exposure of cells to 10 microM trimetrexate for 30 min produced approximately 45% interconversion of tetrahydrofolate cofactors to dihydrofolate in the cytosolic fraction, a level much greater than that observed in whole cell extracts (25-30%), but had no effect on folate pools in the crude mitochondrial fraction. These data indicate that subcellular compartmentation accounts, in part, for the failure to oxidize tetrahydrofolate cofactors to dihydrofolate in the presence of antifolate levels that abolish dihydrofolate reductase activity.     

High-affinity binding of [3H]6-nitroquipazine to 5-hydroxytryptamine transporter in human platelets

The characteristics of the binding [3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37 degrees C. The presence of a single saturable high-affinity binding component for [3H]6-nitroquipazine was demonstrated Non-specific binding was estimated in the presence of 1 microM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 0.450 +/- 0.04 nM and a maximal number of binding sites (Bmax) of 2508 +/- 360 fmol/mg protein (mean +/- S.D., n = 4). The kinetically derived dissociation constant (Kd) was 0.431 nM. [3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [3H]5-HT uptake into synaptosome. Moreover, Ki values for drug inhibition of [3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37 degrees C. The present results suggest that the binding sites for [3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.     

Effect of N8-acetylspermidine deacetylase inhibition on the growth of L1210 cells

A selective inhibitor of N8-acetylspermidine deacetylase has been employed to study the role of N8-acetylspermidine deacetylation in the regulation of L1210 cell growth. This inhibitor, 7-[N-(3-aminopropyl) amino] heptan-2-one (APAH), was found to stimulate the growth of L1210 cells at concentrations between 10 microM and 0.5 mM. Maximum stimulation was seen at 100 microM, resulting in significantly increased rates of cell division and maximum cell density. N8-Acetylspermidine levels in L1210 cells were shown to increase significantly after the APAH treatment as would be expected for deacetylase inhibition. The effects of deacetylase inhibition were mimicked by addition of N8-acetylspermidine to the culture medium at concentrations greater than 1 mM as indicated by a subsequent increase in rate of cell growth and maximum cell density. The magnitudes of the increases in growth observed were not large, but this might be expected in cells that are already in a rapid growth phase. Other exogenously added polyamines including N1-acetylspermidine, spermidine, putrescine, and spermine did not stimulate cell growth. These data suggest that stimulation of cell growth occurs as a consequence of N8-acetylspermidine accumulation and N8-acetylspermidine deacetylase inhibition.     

Depletion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate by methotrexate in cultured hepatoma cells.

The effect of the inhibition of dihydrofolate reductase by methotrexate on the cellular folates involved in de novo purine and thymidylate biosynthesis has been measured in H35 hepatoma cells grown in 4 microM folic acid or 20 nM folinic acid. The major cellular folate species in cells from medium with folate or folinate is 10-formyltetrahydrofolate (approximately 5 microM), with lesser amounts of 5,10-methylenetetrahydrofolate and tetrahydrofolate. Cultures were exposed to a pulse dose of methotrexate, resulting in the accumulation of nearly exclusively methotrexate polyglutamates (predominantly Glu3, Glu4, and Glu5), or a continuous exposure to the poorly glutamylated analog threo-4-fluoromethotrexate, resulting in 93% intracellular monoglutamate. At 4 hr and 18 hr after exposure to either compound there was extensive depletion of the reduced folate coenzymes, which generally corresponded to the extent of inhibition of glycine and deoxyuridine incorporation. This was accompanied by an increase of the cellular dihydrofolate and 10-formyldihydrofolate. In the H35 cells the effect of methotrexate polyglutamates on the reduced folate coenzyme pools was restricted to dividing cultures, because the reduced folate coenzymes were not depleted in confluent cultures. The results demonstrate that the methotrexate and methotrexate polyglutamates that initially accumulate within dividing H35 cells readily inhibit dihydrofolate reductase but are not adequate to inhibit thymidylate synthase and prevent the depletion of reduced folate coenzymes. Thus, inhibition of de novo glycine and deoxyuridine incorporation into DNA as a result of dihydrofolate reductase inhibitors appears to be closely related to a reduction in the intracellular concentration of 10-formyltetrahydrofolate and 5,10-methylenetetrahydrofolate, the respective folate coenzymes for de novo purine and thymidylate synthesis.     

Role of deoxycytidine kinase in the inhibitory activity of 5-substituted 2'-deoxycytidines and cytosine arabinosides on tumor cell growth

A number of 5-substituted derivatives of dCyd and 1-beta-D-arabinofuranosylcytosine (araC) have been evaluated for their inhibitory effects on the growth of three murine leukemia cell lines (L1210/0, L1210/BdUrd, and L1210/araC). The L1210/BdUrd and L1210/araC cell lines were selected from the parental L1210/0 cell line by their ability to grow at high concentrations of 5-bromo-2'-deoxyuridine and araC, respectively; the L1210/BdUrd cell line was deficient in dThd kinase activity, whereas the L1210/araC cell was deficient in dCyd kinase activity. The most effective inhibitors of L1210/0 cell proliferation were 5-fluoro-dCyd, araC, and 5-fluoro-araC. Their 50% inhibitory dose fell within the 0.001-0.015 micrograms/ml range. The 5-substituted araC analogues were much less inhibitory for L1210/araC cells but equally inhibitory for L1210/BdUrd as for the parental L1210/0 cell line. The role of dCyd kinase in the antitumor activity of the dCyd and araC analogues was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and araC analogues caused a competitive inhibition of dCyd kinase, the most potent inhibitor being 5-fluoro-dCyd (Ki/Km value 0.24). The Km of dCyd kinase from L1210/0 cells for dCyd was 23.1 microM as compared with 50 microM for araC. These values were increased to 53 and 182 microM, respectively, for the dCyd kinase isolated from L1210/araC cells.     

Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives

A series of N-hydroxy-N'-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCH=NNHC(=NH)NHOH-tosylate] studied could be grouped as: (1) hydroxybenzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2'-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 +/- 5 and 13 +/- 4 microM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihydroxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohydroxybenzylidene-HAGs. 2'-Hydroxy-4'-methoxybenzylidene-HAG was much more active than 2',4'-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitrobenzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2',3',4'- and 3',4',5'-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 microM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 microM respectively) and ribonucleotide reductase activity (122 and 188 microM respectively). The studies of nucleic acid synthesis in L1210 cells using [3H]cytidine as a precursor showed that 2',3',4'-trihydroxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 microM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 microM for 50% inhibition respectively). These results indicate that 2',3',4'-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.     

Binding of [3H]apomorphine to an aporphine binding site as well as to dopamine sites in tissue from bovine caudate nucleus

Binding of the tritiated dopamine (DA) agonists, apomorphine (APO) and a dihydroxyaminotetralin (ADTN) to a membrane preparation from the caudate nucleus of calf brain was compared. Binding of [3H]dihydroxyaminotetralin at small (nM) concentrations followed simple, monophasic inhibition (over 80% at less than 500 nM) by concentrations of apomorphine between 50 pM and 1 mM. Inhibition of the binding of [3H]apomorphine by dihydroxyaminotetralin was more complex, and included in component with a low (microM) affinity for dihydroxyaminotetralin accounting for approx. 20% of total binding. The kinetics of binding of the ligands to high-affinity sites were virtually identical (apparent Kd = 0.81 nM; Bmax = 211 fmol/mg protein) and could not be distinguished by curve-fitting techniques adapted to analysis by microcomputer. In contrast, the binding of [3H]apomorphine with a "blank" defined by excess (10 microM) dihydroxyaminotetralin could be resolved into the same high-affinity component and a lower-affinity site (Kd = 124 nM; Bmax = 5740 fmol/mg). The pharmacology of the lower-affinity binding of [3H]apomorphine was evaluated by coincubating with 0.5 microM dihydroxyaminotetralin to "mask" high-affinity sites, and was compared to high-affinity binding of [3H]apomorphine and [3H]dihydroxyaminotetralin. The high-affinity binding was stereoselective for DA receptor agonists and antagonists. The pharmacology of the lower-affinity site resembled no known DA receptor type and showed highest affinities for aporphines but was not stereoselective and reacted weakly and nonspecifically with dihydroxyaminotetralin, DA, other catecholamines and neuroleptics. Thus, [3H]apomorphine, under certain conditions, may detect an aporphine binding site of uncertain pharmacological significance, as well as high-affinity DA agonist (D-3) sites.     

Characterization of diadenosine tetraphosphate (Ap4A) binding sites in cultured chromaffin cells: evidence for a P2y site.

1. Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A Kd value of 8 +/- 0.65 x 10(-11) M and Bmax value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A Kd value of 5.6 +/- 0.53 x 10(-9) M and a Bmax value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2. The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [3H]-Ap4A from the two binding sites, the Ki values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5 microM, 0.13 microM, 0.062 microM and 0.75 microM for the second binding site. 3. The ATP analogues displaced [3H]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) greater than 5'-adenylyl imidodiphosphate (AMP-PNP) greater than alpha, beta-methylene ATP (alpha, beta-MeATP), in both binding sites. The Ki values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125 microM, 0.5 microM and 0.9 microM for the second binding site.     

Inhibitory effects of prostaglandin A1 on membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters.

Studies are reported that describe the multifaceted inhibitory effects of prostaglandin A1 (PGA1) on processes that govern the transport of folates across the plasma membrane of Chinese hamster ovary (CHO) cells: the reduced folate carrier, RFC1, and ATP-dependent exporters. PGA1 was a noncompetitive inhibitor of MTX influx mediated by RFC1 with a Ki of approximately 21 microM. The onset of inhibition was virtually instantaneous, not reversible, and appeared to require the incorporation of PGA1 into the lipid membrane; surface adsorption alone was insufficient for inhibition of RFC1 transport activity. In contrast, the effect of PGA1 on folic acid transport was small (approximately 20% inhibition of total influx), consistent with the observation that the major portion of folic acid transport in CHO cells is mediated by a low pH mechanism distinct from RFC1. PGA1 was also a potent inhibitor of the ATP-driven efflux of both MTX and folic acid. At a concentration of 7 microM PGA1, the efflux rate constants for these folates were depressed by approximately 70 and approximately 50%, respectively. The net effects of PGA1 on the bidirectional folate fluxes translated into marked alterations in net transport. The addition of 7 microM PGA1 to cells at steady state with 1 microM MTX produced a rapid onset of net uptake and the achievement of an approximately 3-fold increase in the steady-state free MTX level as compared with untreated CHO cells. The addition of 7 microM PGA1 to cells at steady state with 1 microM folic acid produced an approximately 5-fold increase in the free folate level. These studies establish PGA1 as a potent inhibitor of both the reduced folate carrier and ATP-driven folate exporter(s). The noncompetitive nature of the inhibition of RFC1 is unique among anionic compounds, which are usually competitive inhibitors of the carrier.     

Inhibition of inositol phospholipid hydrolysis in endothelial cells by pentobarbital

Barbiturates alter cardiovascular function, in part by an effect on vascular cells. However, a biochemical mechanism for the effect is unknown. We have, therefore, studied the effect of barbiturates on inositol phospholipid hydrolysis in cultured rat aortic endothelial cells. Hydrolysis was stimulated by angiotensin II, norepinephrine and phenylephrine. Pentobarbital, and other barbiturates, inhibited hydrolysis at pharmacological and clinical concentrations (0.1-0.5 mM). The inhibition by pentobarbital was concentration-dependent, reversed by washing, and was decreased by high concentrations of angiotensin II. Kinetic studies gave an apparent Km of hydrolysis by angiotensin II of 1.2 nM, which showed mixed inhibition by pentobarbital (Ki = 0.45 mM). Schild analysis of data obtained from pentobarbital inhibition curves also showed a deviation from a competitive type inhibition. [125I]Angiotensin II was bound to a high-affinity receptor (Kd = 1.2 nM), which showed a competitive type inhibition of binding by pentobarbital (0.5 mM). Although inhibition of [125I]angiotensin II binding appeared to be competitively inhibited by pentobarbital, the data, taken together, point to a deviation from a simple competitive type inhibition.     

The effect of folate on the methotrexate/indomethacin interaction in a murine cancer cell line.

1. The effect of folate on the interaction between methotrexate (a folate analogue) and indomethacin has been examined in murine NC carcinoma cells. 2. Conditioning of NC cells to a physiological (20 nM) folate concentration after culture in a high folate concentration increased the response to methotrexate. The sensitivity of these conditioned cells to methotrexate related inversely to the folate concentration. 3. At 20 nM and 2 microM folate, indomethacin 1 micrograms ml-1 potentiated the cytotoxicity of methotrexate 4 and 8 ng ml-1 (both P < 0.03). 4. When NC cells were incubated with [3H]-methotrexate at 20 nM and 2 microM folate, there was a trend for increased tritium accumulation with indomethacin 0.36 micrograms ml-1 (1 microM; P < 0.01). 5. We conclude that the folate concentration can affect the sensitivity of NC cells to methotrexate, although the degree of potentiation of cytotoxicity by indomethacin remains similar.